Urinary microRNAs can predict response to abiraterone acetate in castration resistant prostate cancer: A pilot study.

BACKGROUND: Despite novel agents have been introduced to treat castration resistant prostate cancer (CRPC) during the last decade, up to one-third of CRPC patients face primary resistance to new generation compounds. Therefore, sensitive molecular tools are urgently needed for reliable treatment selection and response prediction. This study aimed to evaluate urinary miRNAs and blood circulating androgen receptor (AR) transcript level as a tool for noninvasive outcome prediction for CRPC patients undergoing abiraterone acetate (AA) therapy. METHODS: Prostate cancer-specific miR-148a, -365, -375, and -429 were analyzed in 129 urine samples collected from 100 CRPC patients before and during AA therapy via quantitative reverse transcription PCR. To test the prognostic value, urinary miRNA levels alone, as well as combined with AR level were associated with progression-free survival (PFS) and overall survival (OS). RESULTS: Level of urinary miR-375 was the highest in CRPC in comparison to noncancerous controls, as well as in combination with miR-429 was predictive for short PFS in AA-treated patients (HR = 2.2, 95% CI: 1.1-4.2, p = 0.023). Especially high prognostic power of all analyzed miRNAs was observed in CRPC cases with high blood AR levels. For PFS prediction a tandem of miR-429 and high AR reached HR of 5.0 (95% CI: 2.2-11.8, p < 0.001), while for prediction of OS the best combination was demonstrated by miR-148a and AR with HR of 3.1 (95% CI: 1.4-7.1, p = 0.006). CONCLUSIONS: Urinary miRNAs could be used as prognostic biomarkers for CRPC patients to predict response to AA therapy, especially for the cases with high blood AR levels.

Analysis of AR-FL and AR-V1 in Whole Blood of Patients with Castration Resistant Prostate Cancer as a Tool for Predicting Response to Abiraterone Acetate.

PURPOSE: Reliable molecular diagnostic tools are still unavailable for making informed treatment decisions and monitoring the response in patients with castration resistant prostate cancer. We evaluated the significance of whole blood circulating androgen receptor transcripts of full length (AR-FL) and splice variants (AR-V1, AR-V3 and AR-V7) as biomarkers of abiraterone acetate treatment resistance in patients with castration resistant prostate cancer. MATERIALS AND METHODS: After retrospective analysis in 112 prostate specimens AR-FL, AR-V1, AR-V3 and AR-V7 were evaluated in 185 serial blood samples, prospectively collected from 102 patients with castration resistant prostate cancer before and during abiraterone acetate therapy via reverse transcription quantitative polymerase chain reaction. RESULTS: AR-FL was present in all samples while AR-V1, AR-V3, AR-V7 and at least 1 of them was detected in 17%, 55%, 65% and 81% of castration resistant prostate cancer blood samples, respectively. The highest amount of AR-V1 was found in blood of patients whose response time was short and medium in comparison to extended. Patients with a higher level of AR-FL and/or AR-V1 had the shortest progression-free survival and overall survival (p <0.0001). CONCLUSIONS: Blood circulating AR-FL or AR-V1 can serve as blood based biomarkers for identification of the primary resistance to abiraterone acetate and the tool to monitor de novo resistance development during abiraterone acetate treatment.

The utility of urine-circulating miRNAs for detection of prostate cancer.

BACKGROUND: In this paper, the utility of urine-circulating microRNAs (miRNAs) as the potential biomarker of prostate cancer (PCa), the second most prevalent male cancer worldwide, was evaluated. METHODS: Cancerous (N=56) and non-cancerous (N=16) prostate tissues were analysed on TaqMan Low Density Array, with the initial screening of 754 miRNAs in a subset of the samples. The abundance of selected miRNAs was analysed in urine specimens from two independent cohorts of patients with PCa (N=215 overall), benign prostatic hyperplasia (BPH; N=23), and asymptomatic controls (ASC; N=62) by means of quantitative reverse transcription PCR. RESULTS: Over 100 miRNAs were found deregulated in PCa as compared with non-cancerous prostate tissue. After thorough validation, four miRNAs were selected for the analysis in urine specimens. The abundance of miR-148a and miR-375 in urine was identified as specific biomarkers of PCa in both cohorts. Combined analysis of urine-circulating miR-148a and miR-375 was highly sensitive and specific for PCa in both cohorts (AUC=0.79 and 0.84) and strongly improved the diagnostic power of the PSA test (AUC=0.85, cohort PCa1), including the grey diagnostic zone (AUC=0.90). CONCLUSIONS: Quantitative measurement of urine-circulating miR-148a and miR-375 can serve as the non-invasive tool for sensitive and specific detection of PCa.

Prognostic value of RASSF1 promoter methylation in prostate cancer.

PURPOSE: Patients with prostate cancer who have biochemical recurrence after curative therapy are at higher risk for distant metastasis and cancer specific death. Assessment of aberrant DNA methylation in urine might complement currently used clinical prognostic factors and serve as a noninvasive tool for early prediction of biochemical recurrence after radical prostatectomy. MATERIALS AND METHODS: Promoter methylation of 7 genes was evaluated by methylation sensitive polymerase chain reaction in 149 prostate cancer tissues, 37 noncancerous prostate tissues and 17 benign prostatic hyperplasia samples. Quantitative polymerase chain reaction was used for DNA methylation analysis of the urine of 253 patients with prostate cancer and 32 with benign prostatic hyperplasia. RESULTS: In prostate cancer tissue the most frequently methylated genes were RASSF1, GSTP1 and RARB, which combined were positively identified in 85% of cases. These genes were also methylated in the urine of 60% of patients with prostate cancer. RASSF1 was methylated in 45% of prostate cancer urine samples with methylation intensity significantly higher in prostate cancer than in benign prostatic hyperplasia cases (p = 0.018). In a univariate model RASSF1 methylation and the total number of methylated genes in prostate cancer tissue were predictive of time to biochemical recurrence (p = 0.019 and 0.043, respectively). On multivariate analysis RASSF1 methylation together with pathological stage was the most significant predictor of biochemical recurrence in patients with Gleason score 6 tumors when analyzed in tissue and urine (p ≤0.001). CONCLUSIONS: Hypermethylation of RASSF1 in cancerous tissue and urine from patients with prostate cancer correlated with biochemical recurrence after radical prostatectomy. The prognostic potential of this biomarker deserves further investigation.

Evaluation of the biological activity of naturally occurring 5,8-dihydroxycoumarin.

5,8-Dihydroxycoumarin (5,8-DHC) was isolated from aerial parts of sweet grass (Hierochloë odorata L.) and screened for antioxidant and genotoxic activities. A clear linear dependency of radical scavenging capacity in DPPH• and ABTS•+ assays was determined. 5,8-DHC was very efficient in retarding rapeseed oil oxidation (Oxipress test). TPC (total phenols content) and FRAP (the ability to reduce ferric ion to ferrous ion) assays revealed a somewhat lower antioxidant capacity of 5,8-DHC as compared with gallic acid. Genotoxic activity was tested using different genetic end-points: chromosome aberrations (CAs) and micronuclei (MN) in Wistar rat bone marrow in vivo, CAs and sister chromatid exchanges (SCEs) in human lymphocytes in vitro, and somatic mutations and recombination in Drosophila melanogaster wing cells in vivo. 5,8-DHC did not increase frequency of CAs in rat bone marrow cells, but induced a significant increase of MN. It was slightly mutagenic in the Drosophila melanogaster assay after 120 h of treatment, but not after 48 h of treatment. 5,8-DHC induced both CAs and SCEs in vitro in human lymphocytes in a clear dose-dependent manner. Thus, 5,8-DHC may be classified as weakly genotoxic both in vivo and in vitro.

Combined analysis of TMPRSS2-ERG and TERT for improved prognosis of biochemical recurrence in prostate cancer.

Prostate cancer (PCa) is a heterogeneous disease with diverse clinical outcomes. TMPRSS2-ERG is the most common gene fusion in PCa, whereas activation of telomerase is a common feature of various malignancies. The aim of our study was to explore the combined utility of these and some other biomarkers in predicting biochemical recurrence after radical prostatectomy. Prostate specimens and urine sediments from 179 previously untreated patients with pT2-pT3 stage PCa were analyzed for expression of telomerase (TERT and TR) and the TMPRSS2-ERG fusion gene by means of reverse transcription PCR. Real-time PCR was used for quantification of ERG and SPINK1 expression. In total, 74% (117/158) of the prostate adenocarcinomas were positive for the TMPRSS2-ERG and/or TERT expression. Noninvasively, these transcripts were identified in 31% (19/61) of catheterized urine specimens. Significantly higher expression of ERG was detected in TMPRSS2-ERG-positive tumors (P<0.0001), whereas more intense expression of SPINK1 was characteristic for the TMPRSS2-ERG-negative tumors (P=0.003). TERT-positive cases also had elevated levels of ERG (P=0.016), suggesting a possible link between aberrant expression of ERG and reactivation of TERT in prostate tumors. The cases negative for both transcripts, TMPRSS2-ERG and TERT, rarely recurred (P=0.014) and showed significantly longer biochemical recurrence-free period (P=0.022) as compared to the TMPRSS2-ERG and/or TERT-positive cases. The results of our study suggest that combined analysis of TMPRSS2-ERG and TERT expression can be a valuable tool for early prediction of biochemical recurrence of PCa after radical prostatectomy.

Influence of Body Mass Index and Duration of Disease on Chromosome Damage in Lymphocytes of Patients with Diabetes.

It is well-established that patients with diabetes mellitus (DM) have a higher incidence of several types of cancer. The precise mechanisms of this association are still unknown, but obesity and chronic inflammation-induced reactive oxygen species (ROS) are thought to be the main risk factors. ROS may produce different DNA damage, which could eventually lead to cancer. The main objective of this study was to evaluate the relation of chromosome aberrations (CA) with disease status, demographics, and clinical parameters in 33 subjects with type 1 DM (T1DM), 22 subjects with type 2 DM (T2DM), and 21 controls. CAs were analyzed in cultured peripheral blood lymphocytes and subdivided into chromatid (CTA)- and chromosome (CSA)-type aberrations. Compared with controls, higher levels of CTAs and CSAs were observed in T1DM (p = 0.0053 and p = 0.0203, respectively) and T2DM (p = 0.0133 and p = 0.00002, respectively). While there was no difference in CTAs between T1DM and T2DM, CSAs were higher in T2DM (p = 0.0173). A significant positive association between CTAs and disease duration (rs = 0.2938, p = 0.0099) and between CSAs and disease duration (rs = 0.4306, p = 0.0001), age (rs = 0.3932, p = 0.0004), and body mass index (BMI) (rs = 0.3502, p = 0.0019) was revealed. After multiple regression analysis, duration of disease remained significant for CTA, CSA, and CAs (p = 0.0042, p = 0.00003, and p = 0.00002, respectively). For CSA, BMI and the use of statins were the other important confounding variables (p = 0.0105 and p = 0.0763). Thus, this study demonstrated that both T1DM and T2DM patients had a higher number of all types of aberrations than controls, which increases with the prolonged disease duration. Higher BMI was associated with a higher frequency of CSA. The use of statins might be beneficial for reducing chromosome damage, but further investigations are needed to confirm this association.

Frequent methylation of RASSF1 and RARB in urine sediments from patients with early stage prostate cancer.

BACKGROUND. Prostate cancer (PCa) is the second most prevalent malignancy among males, characterized by high mortality rates. Aberrant DNA methylation in promoters of tumor suppressor genes is an early and frequent event during prostate carcinogenesis. Modern techniques allow a sensitive detection of DNA methylation biomarkers in bodily fluids from cancer patients offering a noninvasive tool for PCa monitoring. Our study aimed at the analysis of DNA methylation in urine sediments from PCa patients for the selection of most informative noninvasive biomarkers. MATERIAL AND METHODS. Real-time methylation-specific polymerase chain reaction was used for the detection of methylated RASSF1, RARB, and GSTP1 genes in catheterized urine specimens from 34 patients with biopsy-proven early or medium stage PCa. RESULTS. At least one gene was methylated in urine sediments from 28 cases with PCa, with a sensitivity of the test reaching 82%. RASSF1 was methylated in 71% (24 of 34), RARB in 44% (15 of 34), and GSTP1 in 3% (1 of 34) of the specimens. High level of methylation (≥50%) in RARB and RASSF1 genes was detected in 40% and 20% of cases, respectively. A significant association was observed between high level of RARB methylation and Gleason score (P=0.01), while methylation of at least one gene occurred more frequently in urine DNA of older patients (P=0.02). CONCLUSIONS. Results of our study show a high sensitivity of DNA methylation biomarkers, especially RASSF1 and RARB, for the early and noninvasive detection of PCa.

Decreased expression of MT1E is a potential biomarker of prostate cancer progression.

Differentiation of indolent and aggressive prostate carcinoma (PCa) at the time of diagnosis is currently one of the major challenges. This study aimed at identification of prognostic biomarkers to aid in predicting biochemical recurrence (BCR) of the disease. Microarray-based gene expression profiling in tissues of 8 BCR and 8 No-BCR cases revealed expression differences of 455 genes, most of which were down-regulated in BCR cases. Eleven genes were selected for validation by real-time PCR in the first PCa cohort (N = 55), while seven of them were further validated in the second, independent, PCa cohort (N = 53). Down-regulation of MT1E (p < 0.001) and GPR52 (p = 0.002) expression and up-regulated levels of EZH2 (p = 0.025) were specific biomarkers of BCR in at least one of the two PCa cohorts, but only MT1E expression retained the independent prognostic value in a multivariate analysis (p < 0.001). DNA methylation analysis (114 PCa and 24 non-cancerous tissues) showed frequent MT1E methylation in PCa (p < 0.001) and was associated (p < 0.010) with the down-regulated expression in one PCa cohort. The results of our study suggest MT1E down-regulation as a potential feature of aggressive PCa.

Heterogeneity of DNA methylation in multifocal prostate cancer.

Most prostate cancer (PCa) cases are multifocal, and separate foci display histological and molecular heterogeneity. DNA hypermethylation is a frequent alteration in PCa, but interfocal heterogeneity of these changes has not been extensively investigated. Ten pairs of foci from multifocal PCa and 15 benign prostatic hyperplasia (BPH) samples were obtained from prostatectomy specimens, resulting altogether in 35 samples. Methylation-specific PCR (MSP) was used to evaluate methylation status of nine tumor suppressor genes (TSGs), and a set of selected TSGs was quantitatively analyzed for methylation intensity by pyrosequencing. Promoter sequences of the RASSF1 and ESR1 genes were methylated in all paired PCa foci, and frequent (≥75 %) DNA methylation was detected in RARB, GSTP1, and ABCB1 genes. MSP revealed different methylation status of at least one gene in separate foci in 8 out of 10 multifocal tumors. The mean methylation level of ESR1, GSTP1, RASSF1, and RARB differed between the paired foci of all PCa cases. The intensity of DNA methylation in these TSGs was significantly higher in PCa cases than in BPH (p < 0.001). Hierarchical cluster analysis revealed a divergent methylation profile of paired PCa foci, while the foci from separate cases with biochemical recurrence showed similar methylation profile and the highest mean levels of DNA methylation. Our findings suggest that PCa tissue is heterogeneous, as between paired foci differences in DNA methylation status were found. Common epigenetic profile of recurrent tumors can be inferred from our data.

Spontaneous and benzo[a]pyrene-induced micronuclei in the embryos of the black-headed gull (Larus ridibundus L.).

The spontaneous levels of micronuclei in erythrocytes were established in embryos of the black-headed gull of two natural populations. In total 216 blood samples from the same number of individuals were examined. A statistically significant decrease in the number of spontaneous micronucleated erythrocytes was found after 13 days of incubation. We found no statistically significant difference in the spontaneous frequencies of micronucleated erythrocytes in the embryos of the two colonies studied, although they differed in anthropogenic load. Results of analysis of variance indicated that egg incubation time was the only variable significantly (P=0.0001) affecting spontaneous frequency of micronucleated erythrocytes in the embryos of black-headed gulls. We also took 78 eggs of different developmental stages from both colonies and exposed them for a further 24h to a dose of benzo[a]pyrene (30 microg per egg). After exposure to benzo[a]pyrene, the frequency of micronucleated erythrocytes was not increased in the embryos incubated for a total period of 13 days. A statistically significant increase in the number of micronucleated erythrocytes was recorded in the benzo[a]pyrene-treated embryos incubated for a total period of 14 days. Decrease in numbers of spontaneous micronucleated erythrocytes after the 13 day of incubation and increased levels of benzo[a]pyrene-induced micronuclei after the 13 day of incubation were discussed to be caused by changes in spleen and liver function in advanced developmental stages of the embryo.

Seasonal variation in the frequency of abnormal anaphases and mitotic index values in wild populations of herb-Paris (Paris quadrifolia L., Trilliaceae): implications for genetic monitoring.

The main aim of our study was to investigate seasonal variation in the frequency of abnormal anaphases and mitotic index values in wild populations of herb-Paris (Paris quadrifolia L., Trilliaceae). Plant material was collected in the year 2000 in Norway and in the year 2001 in Lithuania. There was statistically significant variation in the mitotic index values (chi(2)=1087.9, d.f.=16, P<0.0001) with the highest values during the active growth period in May and the lowest values at the end of vegetation period in September. Seasonal variation in the frequency of abnormal anaphases was statistically significant as well (chi(2)=28.23, d.f.=16, P=0.0297). The most frequent type of anaphase abnormality was vagrant chromosomes (64.2%) followed by bridges (28.6%), fragments (3.6%), sticky chromosomes (2.4%) and multipolar anaphases (1.2%). During the fieldwork, quite deep late frosts occurred. Mitotic index was lower in the plants collected immediately after the frosts or 1 week later than in the plants sampled before the frosts (52+/-13 and 123+/-15, respectively, P=0.0014). On the contrary, frequency of abnormal anaphases was statistically significantly elevated (P=0.0082) in plants after the frosts (6.35+/-1.54%) when compared to plants before the frosts (2.49+/-0.56%). Our results clearly indicated significant variation in the mitotic index values and frequency of abnormal anaphases in the wild populations of herb-Paris during the growth season. This variation may be related to the physiological conditions of the analysed plants as well as to certain ecological factors.

Aberrant p16 promoter methylation in smokers and former smokers with nonsmall cell lung cancer.

Hypermethylation of cytosines in CpG-rich islands of the promoter regions of regulatory genes has been discovered as a common mechanism of gene silencing during carcinogenesis. We analysed 64 primary lung carcinomas for promoter methylation of the tumour suppressor genes (TSGs) p16 (p16(INK4a)/CDKN2A) and p14 (p14(ARF)) by methylation-specific PCR, in order to evaluate aberrant methylation as a potential biomarker for epigenetic alterations in tobacco-related lung cancer. Methylation of p16 was observed in 34% (22/64) of the lung tumours examined. In particular, p16 methylation occurred in nonsmall cell lung cancer (NSCLC) only, with 41 % (22/54) of the tumours being positive. The highest frequency was found in large cell carcinoma (5/7, 71%), followed by adenocarcinoma (9/25, 36%) and squamous cell carcinoma (7/21, 33%). Methylation of the p14 gene was less frequent in lung cancer (4/52, 8%). When association with tobacco smoking was analysed, 42% (21/50) of NSCLC from ever smokers exhibited p16 methylation. Interestingly, the analysis revealed a significantly higher risk of p16 methylation in former smokers as compared to current smokers [odds ratio (OR) 5.1; 95% confidence interval (CI) 1.3-22]. The difference was retained after adjustment for age (OR 3.7; 95% CI 0.9-17). The promoter methylation results were then combined with data on genetic alterations determined previously in the same set of tumours. This data similarly showed that p16 methylation in parallel with p53 gene mutation or p14 methylation occurred more frequently in former smokers than in current smokers (44% vs. 14%; P = 0.035). Taken together, our data suggest that analysis of promoter methylation in TSGs may provide a valuable biomarker for identification of groups with an elevated risk of cancer, such as smokers and ex-smokers.